Inhibitors of protein kinases

ABSTRACT

Protein kinases are regulators of cellular signaling and their functional dysregulation is common in carcinogenesis and many other disease states or disorders. The present invention relates to novel chemical entities that have biological activity to modulate mammalian protein kinase enzymes. In particular, compounds of the invention display potent inhibition of breast tumor related kinase (BRK).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 of PCTInternational Application PCT/US2016/056439 designating the UnitedStates of America, and filed Oct. 11, 2016 the entire contents of whichare hereby incorporated herein by reference. This application claims thebenefit under 35 U.S.C. §119(e) to U.S. Provisional Application No.62/241,903, filed on Oct. 15, 2015, the entire contents of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel chemical entities that havebiological activity to modulate mammalian protein kinase enzymes.

BACKGROUND OF THE INVENTION

The information provided herein is intended solely to assist theunderstanding of the reader. None of the information provided norreferences cited is admitted to the prior art to the present invention.Each of the references cited herein is incorporated in its entirety.

The identification of the molecular events that underlie the developmentof human tumors presents a major challenge in the design of improvedstrategies in the prevention, management, and cure of these diseases(Barker, K. T. et al. BRK Tyrosine Kinase Expression in a HighProportion of Human Breast Carcinomas, Oncogene 1997, 15:799-805). Therole of aberrantly regulated protein tyrosine kinases (PTKs) in humantumor development is the subject of intense investigation (Barker id.).

Protein kinases are regulators of cellular signaling and theirfunctional dysregulation common in carcinogenesis and many other diseasestates or conditions (Mizuguchi, Y. et al., Breast Tumor Kinase/ProteinTyrosine Kinase 6 (Brk/PTK6). J Hepatology 2015, 63, 399-407; Mahmoud,K. A. et al., Discovery of 4-Anilino α-Carbolines as Novel BrkInhibitors. Bioorganic & Medicinal Chemistry Letters 2014, 24:1948-1951:Nobel, M. E. M. Protein Kinase Inhibitors: Insights into Drug Designfrom Structure. Science 2004, 303:1800-1805). The human genome encodesover 500 protein kinases that share a catalytic domain conserved insequence and structure but which are notably different in how theircatalysis is regulated (Nobel id.). Protein kinases regulate key signaltransduction cascades that control or are involved in the control ofphysiological functions including cellular growth and proliferation,cell differentiation, cellular development, cell division, stressresponse, transcription regulation, aberrant mitogenesis, angiogeneisis,abnormal endothelial cell-cell or cell-matrix interactions duringvascular development, inflammation, Jun-N-terminal kinase (JNK) signaltransduction, and several other cellular processes (see U.S. Pat. No.8,470,818). Protein kinase inhibitors have been established as promisingdrugs that inhibit overactive protein kinases in cancer cells (Mahmoudid.).

A partial, non-limiting list of these kinases includes: BRK, FGR,PDGFRα(V561D), DDR2, LYNa, SRM, PDGFRα, LCK, DDR1, KDR, ACK, JAK1, LYNb,KIT, CSK, YES, KIT(V560G), BLK, MST1, JAK2, RET(S891A), SRC, FYN(isoforma), RET(G691S), FYN(isoform b), PDGFRβ, RET, FLT4, RET(Y791F), skMLCK,FRK, MST2, FLT1, AurA, FLT3, JAK3, RET(M918T), WNK3, p388, FGFR2, MNK1,MNK2, PIK3CA/PIK3R1, PDGFRα(D842V), MET, FGFR1, BRAF(V600E), MAP2K5,KIT(D816E), ALK, FGFR3, RAF1, MAP2K3, HER4, KIT(D816V), Erk5, EGFR,YES(T348I), KIT(V654A), KIT(D816Y), PDGFRα(T674I), BRAF, ABL, HER2,EPHA5, ROCK2, KIT(T670I), PKD3, MST4, MAP2K1, MAP2K2, MST3, ROCK1,IGF1R, PKD2, MAP2K6, Erk2, PKD1, MAP2K7, Erk1, MAP2K4, and BTK (see U.S.Pat. No. 8,470,818 B2 for detailed information on kinase nomenclatureand biological properties). Aberrant kinase activity has been observedin many disease states including benign and malignant proliferativedisorders as well as diseases resulting from inappropriate activation ofthe immune and nervous systems.

The novel compounds of this invention inhibit the activity of one ormore protein kinases and are, therefore, expected to be useful in thetreatment of kinase-related diseases or conditions.

SUMMARY OF INVENTION

The present invention concerns compounds active on protein kinases ingeneral.

In one aspect, the present invention provides a compound having formulaI:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula I, R¹ is a substituted lower alkyl or lower alkenyl, whereinthe lower alkyl or lower alkenyl is substituted with one or moresubstituents selected from —OH, ═O, and alkoxy; and R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, and R⁹ are independently selected from the group consisting ofhydrogen, halogen, optionally substituted lower alkyl, optionallysubstituted lower alkenyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, —CN, and —NO₂.

In one aspect, the present invention provides a compound having formulaII:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula II, R¹ is a substituted lower alkyl or lower alkenyl, whereinthe lower alkyl or lower alkenyl is substituted with one or moresubstituents selected from —OH, ═O, and alkoxy; and R⁴, R⁵, and R⁸ areindependently selected from the group consisting of hydrogen, halogen,optionally substituted lower alkyl, optionally substituted loweralkenyl, optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, —CN, and —NO₂.

In one aspect, the present invention provides a compound having formulaIII:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula III, R¹ is a substituted lower alkyl or lower alkenyl,wherein the lower alkyl or lower alkenyl is substituted, with one ormore substituents selected from —OH, ═O, and alkoxy; and R⁴, R⁵, and R¹⁰are independently selected from the group consisting of hydrogen,halogen, optionally substituted lower alkyl, optionally substitutedlower alkenyl, optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, —CN, and —NO₂.

In one aspect, the present invention provides a compound having formulaIV:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula III, R¹ is a substituted lower alkyl or lower alkenyl,wherein the lower alkyl or lower alkenyl is substituted with one or moresubstituents elected from —OH, ═O, and alkoxy.

In one aspect, the present invention provides compound having thestructure of 200-17:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-73:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of formula 200-93:

all salts, prodrugs, tautomers, and the cis and trans isomers (200-93aand 200-93b) thereof.

In another aspect, the present invention provides a compound having thestructure of formula 200-115:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-117:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-123:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-139:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-149:

all salts, prodrugs, tautomers, and isomers thereof.

In one aspect, the invention provides a method for treating a proteinkinase-mediated disease or condition in an animal or human subjectwherein the method involves administering to the subject an effectiveamount of formulas I, II, III, or IV, 200-17, 200-73, 200-93, 200-93a,200-93b, 200-115, 200-117, 200-123, 200-139 or 200-149. The terms“treat,” “therapy,” and like terms refer to the administration ofcompounds in an amount effective to prevent, alleviate, or ameliorateone or more symptoms of a disease condition, i.e., indication, and/or toprolong the survival of the subject being treated. The term “proteinkinase-mediated disease or condition” refers to a disease or conditionin which the biological function of a protein kinase affects thedevelopment, course, and/or symptoms of the disease or condition. Aprotein kinase-mediated disease or condition includes a disease orcondition for which modulation provides a positive effect, i.e., one inwhich treatment with protein kinase inhibitors, including compoundsdescribed herein, provides a therapeutic benefit to the subject with orat risk of the disease or condition.

In one aspect, the invention provides a method for treating a breasttumor related kinase (BRK) mediated disease or condition in a mammal,wherein the method involves administering to the subject an effectiveamount of a compound of the invention. BRK is also known as proteintyrosine kinase 6 (PTK6). The term BRK mediated disease or conditionrefers to a disease or condition in which the biological function ofBRK, including any mutations thereof, affects the development, course,and/or progression of the disease or condition, and/or in whichmodulation of BRK alters the development, course, and/or symptoms of thedisease or condition. BRK includes, but is not limited to, BRK andmutations of BRK.

The compounds of formula I, II, III, or IV, 200-17, 200-73, 200-93,200-93a, 200-93b, 200-115, 200-117, 200-123, 200-139 or 200-149described herein may be administered in an effective amount. An“effective amount” or “therapeutically effective amount” is an amount ofa preparation that alone, or together with further doses, produces thedesired response. Desired response may involve: (1) halting theprogression of the disease or condition, (2) delaying the onset of thedisease or condition, and (3) preventing the disease or condition fromoccurring, although it may also imply only slowing of the disease orcondition.

In reference to compounds of the invention, specification of a compoundor group of compounds includes pharmaceutically acceptable salts,prodrugs(s), and all isomers (cis/trans; enantiomers, and diasteriomers)of such compounds.

In one aspect, the invention provides for pharmaceutical compositionsthat include a therapeutically effective amount of a compound offormulas I, II, II, or IV, 200-17, 200-73, 200-93, 200-93a, 200-93b,200-115, 200-117, 200-123, 200-139 or 200-149 in free form or in apharmaceutically acceptable salt form and at least one pharmaceuticallyacceptable carrier, excipient, and/or diluent.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein the following definitions apply unless clearly indicatedotherwise: By “chemical structure” or “chemical substructure” is meantany definable atom or group of atoms that constitute an individuallyidentifiable portion of a molecule, such as a substituent moiety, a corewhich is optionally substituted, and the like. Normally, chemicalsubstructures of a ligand can have a role in binding of the ligand to atarget molecule, or can influence, the three-dimensional shape,electrostatic charge, and/or conformational properties of the ligand.

The term “prodrug” is a compound that, upon in vivo administration, ismetabolized by one or more steps or processes or otherwise converted tothe biologically, pharmaceutically, or therapeutically active form ofthe compound. To produce a prodrug, the pharmaceutically active compoundis modified such that the active compound will be regenerated bymetabolic processes.

The term “binds” in connection with the interaction between a target anda potential binding compound indicates that the potential bindingcompound associates with the target to a statistically significantdegree as compared to association with proteins generally (i.e.,non-specific binding).

As used herein the term “modulating” or “modulate” refer to an effect ofaltering a biological activity, especially a biological activityassociated with a particular biomolecule such as a protein kinase. Forexample, an agonist or antagonist of a particular biomolecule modulatesthe activity of that biomolecule, e.g., an enzyme, by either increasing(e.g. agonist, activator), or decreasing (e.g. antagonist, inhibitor)its activity. This type of activity is typically indicated in terms ofan inhibitory concentration (IC₅₀) for an inhibitor or an excitationconcentration (EC₅₀) for an activator.

As used herein in connection with compounds of the invention, the term“synthesizing” and like terms means chemical synthesis from one or moreprecursor materials. Further, by “assaying” is meant the creation ofexperimental conditions and the gathering of data regarding a particularresult of the experimental conditions. For example, enzymes can beassayed based on their ability to act upon a detectable substrate. Acompound or ligand can be assayed based on its ability to bind to aparticular target molecule or molecules.

The term “lower alkyl” is art-recognized, and includes saturatedaliphatic groups, including straight-chain alkyl groups andbranched-chain alkyl groups. In certain embodiments, a straight-chain orbranched-chain alkyl has about 6 or fewer carbon atoms in its backbone(e.g., C1-C6 for straight chain, C3-C6 for branched chain).

The term “lower alkenyl” refers to an unsaturated straight or branchedhydrocarbon having at least one carbon-carbon double bond, such as astraight or branched group 2 to 6 carbon atoms, referred to herein asC2-C6 alkenyl.

The term “cycloakyl” refers to a 3 to 7 membered monocyclic ring ofaliphatic groups, including C3-C7, that is optionally substituted withalkyl, alkenyl, alkoxyl, or optionally substituted amino, halogens,cyano (—CN), or nitro (—NO₂).

The term “aryl” alone or in combination refers to a monocyclic orbicyclic ring system containing aromatic hydrocarbons such as phenyl ornaphthyl, which may be optionally fused with a cycloalkyl of preferably5 to 7 carbon atoms, more preferably 5 to 6 carbon atoms.

The term “heterocycloalkyl” refers to a saturated or unsaturated 5non-aromatic cycloalkyl group having from 5 to 10 carbon atoms in whichfrom 1 to 3 carbon atoms in the ring are replaced by heteroatoms of O,S, or N, and are optionally fused with benzo or heteroaryl of 5 to 6ring members.

The term “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxytert-butoxy, and the like.

The term “substituted amines” moiety that may be represented as NR₂where R is independently hydrogen or alkyl.

“Halogen” refers to chloro (Cl), fluoro (F), bromo (Br), or iodo (I).

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures.

The term “pharmaceutically acceptable” means that the indicated materialdoes not have properties that would cause a reasonably prudent medicalpractitioner to avoid administration of the material to a patient,taking into consideration the disease or condition to be treated and therespective route of administration. For example, it is commonly requiredthat such a material be essentially sterile, e.g., for an injectable.

The term “pharmaceutically acceptable salts” refers to salts that arenon-toxic in the amounts and concentrations at which they areadministered. The preparation of such salts can facilitatepharmacological use by altering the physical characteristics of acompound (solubility) without preventing it from exerting itsphysiological effect.

The term “pharmaceutically acceptable composition” refers to apharmaceutically active compound and one or more pharmaceuticallyacceptable carriers, excipients, and/or diluents.

The term “therapeutically effective amount” or “effective amount” is anamount of a preparation that alone, or together with further doses,produces the desired response. This may involve halting the progressionof the disease or condition, delaying the onset of the disease orcondition, or preventing the disease or condition from occurring,although it may also imply only temporarily slowing of the disease orcondition.

The term “protein kinase-mediated disease or condition” refers to adisease or condition in which the biological function of a proteinkinase affects the development, course, and/or symptoms of the diseaseor condition.

The term “mutants” refers to single or multiple amino acid changes in aprotein as compared to the wild-type protein amino acid sequence.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers, orsteps.

Compounds of the Invention

In one aspect, the present invention provides a compound having formulaI:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula I, R¹ is a substituted lower alkyl or lower alkenyl, whereinthe lower alkyl or lower alkenyl is substituted with one or moresubstituents selected from —OH, ═O, and alkoxy; and R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, and R⁹ are independently selected from the group consisting ofhydrogen, halogen, optionally substituted lower alkyl, optionallysubstituted lower alkenyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, —CN, and —NO₂.

In one aspect, the present invention provides a compound having formulaII:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula II, R¹ is a substituted lower alkyl or lower alkenyl, whereinthe lower alkyl or lower alkenyl is substituted with one or moresubstituents selected from —OH, ═O, and alkoxy; and R⁴, R⁵, and R⁸ areindependently selected from the group consisting of hydrogen, halogen,optionally substituted lower alkyl, optionally substituted loweralkenyl, optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, —CN, and —NO₂.

In one aspect, the present invention provides a compound having formulaIII:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula III, R¹ is a substituted lower alkyl or lower alkenyl,wherein the lower alkyl or lower alkenyl is substituted with one or moresubstituents selected from —OH, ═O, and alkoxy; and R⁴, R⁵, and R¹⁰ areindependently selected from the group consisting of hydrogen, halogen,optionally substituted lower alkyl, optionally substituted loweralkenyl, optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, —CN, and —NO₂.

In one aspect, the present invention provides a compound having formulaIV:

all salts, prodrugs, tautomers and isomers thereof. In some embodimentsof formula III, R¹ is a substituted lower alkyl or lower alkenyl,wherein the lower alkyl or lower alkenyl is substituted with one or moresubstituents selected from —OH, ═O, and alkoxy.

In one aspect, the present invention provides a compound having thestructure of 200-17:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-73:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-93 and the resolved isomers 200-93a and 200-93b:

all salts, prodrugs, tautomers, and cis or trans isomers (200-93a and200-93b) thereof.

In another aspect, the present invention provides compound having thestructure of 200-115:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-117:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-123:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-139:

all salts, prodrugs, tautomers, and isomers thereof.

In another aspect, the present invention provides a compound having thestructure of 200-149:

all salts, prodrugs, tautomers, and isomers thereof.

Protein Kinase Targets and Indications of the Invention

Protein kinases play key roles in propagating biochemical signals indiverse biological pathways. More than 500 kinases have been described,and specific kinases have been implicated in a wide range of diseasesconditions. In one aspect, the invention provides methods for treating aprotein kinase-mediated disease or condition in an animal or humansubject, (i.e., indications) such as without limitation, cancer,cardiovascular disease, inflammatory disease, neurological disease, andother diseases. As such, kinases represent important control points forsmall molecule therapeutic intervention.

In another aspect, the invention provides a method for modulating theactivity of a protein kinase selected from the group consisting of BRK,FGR, PDGFRα(V561D), DDR2, LYNa, SRM, PDGFRα, LCK, DDR1, KDR, ACK, JAK1,LYNb, KIT, CSK, YES, KIT(V560G), BLK, MST1, JAK2, RET(S891A), SRC,FYN(isoform a), RET(G691S), FYN(isoform b), PDGFRβ, RET, FLT4,RET(Y791F), skMLCK, FRK, MST2, FLT1, AurA, FLT3, JAK3, RET(M918T), WNK3,p38β, FGFR2, MNK1, MNK2, PIK3CA/PIK3R1, PDGFRα(D842V), MET, FGFR1,BRAF(V600E), MAP2K5, KIT(D816E), ALK, FGFR3, RAF1, MAP2K3, HER4,KIT(D816V), Erk5, EGFR, YES(T348I), KIT(V654A), KIT(D816Y),PDGFRα(T674I), BRAF, ABL, HER2, EPHA5, ROCK2, KIT(T670I), PKD3, MST4,MAP2K1, MAP2K2, MST3, ROCK1, IGF1R, PKD2, MAP2K6, Erk2, PKD1, MAP2K7,Erk1, MAP2K4, and BTK by contacting the protein kinase with an effectiveamount of a compound of formulas I, II, III, IV, 200-17, 200-73, 200-93,200-93a, 200-93b, 200-115, 200-117, 200-123, 200-139 or 200-149.

In another aspect, the invention provides a method for treating aprotein kinase-mediated disease or condition in an animal subject,wherein the method involves administering to the subject an effectiveamount of a composition including a compound of formulas I, II, III, IV,200-17, 200-73, 200-93, 200-93a, 200-93b, 200-115, 200-117, 200-123,200-139 or 200-149.

In one aspect, the invention provides a method for treating a disease orcondition, mediated by a protein kinase selected from the groupconsisting of BRK, FGR, PDGFRα(V561D), DDR2, LYNa, SRM, PDGFRα, LCK,DDR1, KDR, ACK, JAK1, LYNb, KIT, CSK, YES, KIT(V560G), BLK, MST1, JAK2,RET(5891A), SRC, FYN(isoform a), RET(G691S), FYN(isoform b), PDGFRβ,RET, FLT4, RET(Y791F), skMLCK, FRK, MST2, FLT1, AurA, FLT3, JAK3,RET(M918T), WNK3, p38β, FGFR2, MNK1, MNK2, PIK3CA/PIK3R1 PDGFRα(D842V),MET, FGFR1, BRAF(V600E), MAP2K5, KIT(D816E), ALK, FGFR3, RAF1, MAP2K3,HER4, KIT(D816V), Erk5, EGFR, YES(T348I), KIT(V654A), KIT(D816Y),PDGFRα(T674I), BRAF, ABL, HER2, EPHA5, ROCK2, KIT(T670I), PKD3, MST4,MAP2K1, MAP2K2, MST3, ROCK1, IGF1R, PKD2, MAP2K6, Erk2, PKD1, MAP2K7,Erk1, MAP2K4, and BTK by contacting the protein kinase with an effectiveamount of a compound of formulas I, II, III, or IV, 200-17, 200-73,200-93, 200-93a, 200-93b, 200-115, 200-117, 200-123, 200-139 or 200-149.

A number of different assays for kinase activity can be utilized forassaying for active modulators and/or determining specificity of amodulator for a particular kinase or group of kinases. In addition tothe assay mentioned in the Examples below, one of ordinary skill in theart will know of other assays that can be utilized or can be modifiedfor a particular application.

In a commonly used in vitro screen used to measure inhibition of batteryof selected protein kinases (see Example 9) including BRK, FGR,PDGFRα(V561D), DDR2, LYNa, SRM, PDGFRα, LCK, DDR1, KDR, ACK, JAK1, LYNb,KIT, CSK, YES, KIT(V560G), BLK, MST1, JAK2, RET(S891A), SRC, FYN(isoforma), RET(G691S), FYN(isoform b), PDGFRβ, RET, FLT4, RET(Y791F), skMLCK,FRK, MST2, FLT1, AurA, FLT3, JAK3, RET(M918T), WNK3, p38β, FGFR2, MNK1,MNK2, PIK3CA/PIK3R1, PDGFRα(D842V), MET, FGFR1, BRAF(V600E), MAP2K5,KIT(D816E), ALK, FGFR3, RAF1, MAP2K3, HER4, KIT(D816V), Erk5, EGFR,YES(T348I), KIT(V654A), KIT(D816Y), PDGFRα(T674I), BRAF, ABL, HER2,EPHA5, ROCK2, KIT(T670I), PKD3, MST4, MAP2K1, MAP2K2, MST3, ROCK1,IGF1R, PKD2, MAP2K6, Erk2, PKD1, MAP2K7, Erk1, MAP2K4, and BTK,compounds 200-17, 200-73, 200-93a, 200-93b, 200-115, 200-117, 200-123,and 200-139 were found to display activity to inhibit BRK, SRM, FOR,LCK, KIT, and JAK1, among others (see Tables 1 and 2).

In one aspect, compounds 200-17, 200-73, 200-93a, 200-93b, 200-115,200-117, 200-123, and 200-139 displayed low nM activity to greater than30% inhibition of Breast tumor related kinase (BRK), or protein tyrosinekinase 6 (PTK6) BRK. As described below, inhibition of BRK is animportant target for a cancer therapy, particularly breast cancer, asBRK participates in both cell dysregulation and metastasis. BRKinhibition may offer a therapeutic approach for treating patients withHer2 targeted therapy-resistant breast cancers and patients withERBB2/HER2-positive breast cancers (see below).

As a further test of biological activity, compounds were assayed forinhibition of cell growth using the breast cancer cell line T-47D (seeExample 10). In this cell based assay, the IC₅₀ values for 200-17,200-73, 200-93b, 200-115, 200-117, 200-123, 200-139 and 200-149 wereless than 20 μM, while the IC₅₀ value for 200-93a was greater than 20μM.

Protein kinase targets for compounds of formula I, II, III, and IV,200-17, 200-73, 200-93, 200-93a, 200-93b, 200-115, 200-117, 200-123,200-139 and 200-149 include the following: ALK, B-Raf, C-Raf-1, EGFR,Erk2, FGFR, Frk, Fyn, HCK, Her2/Erbb2, Her4/Erbb4, Jak1, Jak2, Jak 3,Kdr, Kit, LCK, MAP2K1, MAP2K2, MAPKAPK2, PDGFR, PDGFRα, PDGFRβ, Ret,Src, TNF-Related Activation-Induced Cytokine (TRANCE), Stk6, Aurora A,Aurora B, Aurora C, and Yes (See U.S. Pat. No. 8,470,818 B2 for detailedinformation about the above listed kinases).

Breast tumor related kinase (BRK), or protein tyrosine kinase 6 (PTK6)is a non-receptor type tyrosine kinase, cloned from a metastatic breasttumor, and overexpressed in a majority of breast cancers (Mizuguchi id.;Ono, H. et al. PTK6 Promotes Cancer Migration and Invasion in PancreaticCancer Cells Dependent on ERK Signaling. 2014, PLoS ONE 9(5): e96060.doi:10.1371/journal.pone.0096060). BRK is composed of an amino-terminalSH3 domain, SH2 domain, and carboxyl-terminal kinase domain (Park, S. H.et al. PTK6 Inhibition Promotes Apoptosis of Lapatinib-Resistant Her2+Breast Cancer Cells by Inducing Bim, Breast Cancer Research 2015, 17:86, doi 10.1186/s13058-015-0594-z). BRK is expressed in many humancancers including breast, ovary, colon head and neck squamous cellcarcinoma, prostate, lung, bladder, pancreas, and gastric, and lymphomas(Mizuguchi id.). BRK is only weakly expressed in normal mammary tissueor benign lesions (Mizuguchi id.). BRK has been shown to be activateddownstream of various receptors in response to stimulation by theirrespective ligands such as EGF, HGF, and IGF (Goel, R. K. et al. Tracingthe Footprints of the Breast Cancer Oncogene BRK—Past Till Present,Biochemica et Biophysics Acta 2015, 1656:39-54). Since the signalingpathways activated by these ligands tend to promote cell proliferationand migration, activation of BRK downstream in these signaling pathwaysis predicated to mechanistically contribute to the promotion ofoncogenesis (Goal id.). Although the physiological function of BRK isdependent on its activation status, level of expression, intracellularlocalization, interaction with various signaling intermediates, andtumor stage or grade, ample evidence suggests that targeting BRK shouldprovide therapeutic benefits in treating breast cancer (Goel id.). Theoncogenic functions of BRK are reported to be mediated via its kinaseactivity, thus the development of clinical inhibitors of the BRK kinasedomain should be an attractive therapeutic target (Goal id.).

In breast cancer, a 20-30% incidence of over expression of the epidermalgrowth factor receptor family tyrosine kinase ERBB2 (HER2, Neu) has beenreported (Peng, M et al. Protein Tyrosine Kinase 6 PromotesERBB2-induced Mammary Gland Tumorigenesis in the Mouse. Cell Death andDisease. 2015, 6:e1848; published online 6 Aug. 2015). Furthermore BRKexpression, activation, and amplification of the BRK gene have beenreported to occur in ERBB2/HER2-positive mammary gland cancers (Pengid.). The role of BRK (PTK6) to promote mammary gland tumorigenesis byactivated ERBB2 was explored by Peng and co-workers using transgenicmice prepared by crossing PTK6 −/− mice with a mouse mammary tumorvirus-ERBB2 transgenic mouse line expressing activated ERBB2. In micelacking BRK, ERBB2-induced tumorigenesis was significantly delayed anddiminished. BRK expression was induced in the mammary glands of ERBB2transgenic mice before tumor development and correlated with activationof signal transducer and activator of transcription 3 (STAT3) andincreased proliferation. Disruption of BRK-impaired STAT3 activation andproliferation. Additionally, phosphorylation of the BRK substrates focaladhesion kinase (FAK) and breast cancer anti-estrogen resistance 1(BCAR1; p130CAS) were decreased in BRK −/− mammary gland tumors. Reducednumbers of metastases were detected in the lungs of BRK −/− miceexpressing activated ERBB2, compared with wild-type ERBB2 transgenicmice. These data support roles for BRK in both ERBB2-induced mammarygland tumor initiation and metastasis, and identify STAT3, FAK, andBCAR1 as physiologically relevant BRK substrates in breast cancer. Thisled the authors to conclude that including BRK inhibitors as part of atreatment regimen could have distinct benefits in ERBB2/HER2-positivebreast cancers.

BRK is highly expressed in Human Epidermal Growth Factor 2+ (Her2+)breast cancers (Park id.). Park and co-workers investigated whether BRKinhibition is an effective strategy to inhibit growth and survival ofHer2+ breast cancer cells, including those that are relatively resistantto Lapatinib, a targeted therapy for Her2+ breast cancer that developedeither intrinsically or was acquired after continuous drug exposure. Theauthors reported that BRK down-regulation induces apoptosis ofLapatinib-resistant Her2(+) breast cancer cells by enhancing Bim, apro-apoptotic Bcl2 family member expressed via p38 activation. As Bimexpression is a critical biomarker for response to many targetedtherapies, BRK inhibition may offer a therapeutic approach to treatingpatients with Her2 targeted therapy-resistant breast cancers.

Recently, Mahmoud and co-workers reported the discovery of derivativesof pyrido[2,3-b]indole derivatives with 4-anilne and 6-substitutions aspotent inhibitors of BRK and/or Her2 and described structure activityrelationships (Mahmoud, K. A. Novel Inhibitors of Breast Cancer RelevantKinases BRK and Her2. Med. Chem. Commun. 2014, 5:659-664). In a dockingmodel developed by these researchers, the ATP-binding pocket in BRK wasfound to favor hydrophilic substituents. Mahmoud and co-workers furtherdisclose a series of 4-anilinoa α-carbolines as novel Brk Inhibitors(Mahmoud, K. Z. et al. “Discovery of 4-Anilino α-Carbolines s Novel Brkinhibitors” Bioorganic and Medicinal Chemistry Letters 2014, 24:1948-1951). In conclusion, the presented series of 4-anilinoα-carbolines turned out as a highly promising class of anticanceragents. The Brk inhibition depends on the kind and positioning of theaniline substituents which lead to nanomolar as well as to inactiveinhibitors. The observed protein kinase inhibition profile documented afirst selectivity of Brk inhibition. The correlation of Brk inhibitionand mediated antiproliferative activity better than that of the reportedlapatinib qualifies the new compound class for further preclinicalstudies (Mahmoud 2014 id.), Zeng et at disclose a group of novelimidazol [1,2a] pyrazin-8 amines as potent inhibitors of BRK and acomputational model for inhibition of BRK (Zing, H. et at “Discovery ofNovel Imidazo [1,2-a] Pyrazin-8-Amines as Brk/PTK6 inhibitors”Bioorganic and Medicinal Chemistry Letters 2011, 21: 5870-5876). Inaddition, Several inhibitors, with single-digit nanomolar targetengagement cell-based activity and an appealing overall DMPK profile,could be used as tool compounds to further validate Brk/PTK6 as apotential target for cancer treatment. None of these compounds have thechemical structure of formulas I, II, III, or IV, 200-17, 200-73,200-93, 200-93a, 200-93b, 200-115, 200-117, 200-123, 200-139, or200-149.

Ren, P. et al. disclose a series of compounds and compositions asprotein kinase inhibitors including BRK inhibitors (US 2010/0048552 A1).The novel compounds inhibit one or more protein kinases and are,therefore, expected to be useful in treatment of kinase-mediateddiseases or conditions. None of these compounds have the chemicalstructure of formulas I, II, III, or IV, 200-17, 200-73, 200-93,200-93a, 200-93b, 200-115, 200-117, 200-123, 200-139, or 200-149.

In one aspect, compounds of formulas I, II, III, or IV, 200-17, 200-73,200-93, 200-93a, 200-93b, 200-115, 200-117, 200-123, 200-139 or 200-149including salts, prodrugs, and/or isomers thereof, can be used inpreparation of medicaments for the treatment of a BRK-mediated diseaseor condition. In particular the disease or condition is cancer. Thetypes of cancers include breast, ovary and colon, and head and necksquamous cell carcinoma. In particular, the disease or condition isbreast cancer including ERBB2/HER2-positive breast cancer and Her2targeted therapy-resistant breast cancer.

The amounts of compounds of formulas I, II, III, or IV, 200-17, 200-73,200-93, 200-93a, 200-93b, 200-115, 200-117, 200-123, 200-139 or 200-149to be administered can be determined by standard procedures taking intoaccount factors such as the compounds IC₅₀; the biological half-life ofthe compound; the age, size, and weight of the subject; and thecondition associated with the subject. In general, routineexperimentation in clinical trials will determine specific ranges foroptimal therapeutic effect for each therapeutic agent and eachadministrative protocol and administration to specific patients will beadjusted to within effective and safe ranges depending on the patient'scondition and responsiveness to initial administration. However, theultimate administration protocol will be regulated according to thejudgment of the attending clinician considering such factors as age,gender, condition, and size of the patient. Generally, doses of activecompounds may range from about 0.01 mg/kg per day to, about 1000 mg/kgper day. Compounds described herein can be administered in single ormultiple doses.

Combination Therapy

In one aspect, the composition to be administered can include aplurality of different pharmacologically active compounds which caninclude a plurality of compounds of the invention including compounds offormulas I, II, III, and IV, 200-17, 200-73, 200-93, 200-93a, 200-93b,200-115, 200-117, 200-123, 200-139 and 200-149. Compounds of formulas I,II, III, and IV, 200-17, 200-73, 200-93, 200-93a, 200-93b, 200-115,200-17, 200-200-123, 200-139 and 200-149 that are therapeuticallyeffective for the same protein kinase-mediated disease or condition,wherein the compounds have an additive or a synergistic effect on thedisease indication, may be found to be effective.

In one aspect, the invention provides methods for treating aBRK-mediated disease or condition in an animal or human subject, whereinthe method involves administering to the subject an effective amount ofa compound of formulas I, II, III, or IV, 200-17, 200-73, 200-93,200-93a, 200-93b, 200-115, 200-117, 200-200-123, 200-139 or 200-149, incombination with one or more other therapies for treating the samedisease or condition. Other therapies include medical procedures (suchas surgeries), therapeutics, and/or radiation. Combination therapy caninclude administration of the compounds described herein with one ormore other therapeutics at different times, or co-administration of thecompounds described herein with one or more other therapeutics. In someembodiments, dosages may be modified for one, or more of the compoundsof the invention or other therapeutics used in combination, suchmodifications being a reduction in the dose amounts relative to acompound or therapy used alone.

It is understood that use in combination includes use with other medicalprocedures, therapeutics, and therapies where the other therapy or drugmay be administered at different times, within a short time period, suchas within 1, 2, 3, or 4-24 hours, or within a longer time period, such a1-2 days, 2-4 days, 4-7 days, or 1-4 weeks. Use of the compounds of theinvention can be in combination with a medical procedure such assurgery, performed on the subject once or infrequently, where thecompounds are administered within a short time or longer time before orafter the medical procedure.

Administration

The methods and compounds will typically be used in therapy for humansubjects with a kinase-mediated disease or condition. However, they mayalso be used to treat similar or identical indications in other animalsubjects. In this context, the terms “subject,” “animal subject,” andthe like refer to human and non-human vertebrates, i.e., mammals, suchas non-human primates, sports and commercial animals, e.g., equines,bovines, porcines, ovines, rodents, and pets, e.g. canines and felines.

In another aspect, the compounds of formulas I, II, III, or IV, 200-17,200-73, 200-93, 200-93a, 200-93b, 200-115, 200-117, 200-200-123, 200-139or 200-149 may be administered intravenously, intramuscularly,subcutaneously, orally, transdermally, transmucosal, rectally, or byinhalation. In the case of intravenous administration, the dose may beadministered as a bolus or infusion.

Pharmaceutical preparations for oral use can be obtained, for example,by combining the compounds of formulas I, II, III, or IV, 200-17,200-73, 200-93, 200-93a, 200-93b, 200-115, 200-117, 200-200-123, 200-139or 200-149 with solid excipients, optionally grinding a resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose (CMC), and/or polyvinylpyrrolidone (PVP:povidone). If desired, disintegrating agents may be added, such as thecross-linked polyvinylpyrrolidone, agar, or alginic acid, or a saltthereof such as sodium alginate.

For injection, the compounds of formulas I, II, III, or IV, 200-17,200-73, 200-93, 200-93a, 200-93b, 200-115, 200-117, 200-200-123, 200-139or 200-149 are formulated in sterile liquid solutions, preferably inphysically compatible buffers or solutions, such as saline solution,Hank's solution, or Ringer's solution. In addition, the compounds may beformulated in solid form and re-dissolved or suspended immediately priorto use. Lyophilized forms can also be produced.

The administration of the compounds described herein can occursimultaneously or sequentially with chemotherapy or radiation. It isunderstood that administration of other therapeutics or drugs to treat amedical disease or condition can be by a different route ofadministration or by the same route of administration.

In another aspect, the use in combination therapy for any route ofadministration includes delivery of compounds of the invention and oneor more other drug therapeutics delivered by the same route ofadministration together in any formulation, or administered together,within an hour, 2 hours, 3 hours, up to 24 hours, in separateformulations or by different routes of administration.

The invention also provides for a pharmaceutical combination, e.g., akit, comprising (a) a first agent which is a compound of the inventionas disclosed herein, in free form or in pharmaceutically acceptable saltform, and (b) at least one co-agent. The kit can include instructionsfor its administration.

General Synthetic Methods

The present invention also includes processes for the preparation ofcompounds of the invention. In the reactions described, it can benecessary to protect reactive functional groups, for example hydroxy,amino, imino, thio, or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.Conventional protecting groups can be used in accordance with standardpractice, for example, see T. W. Greene and P. G. M. Wuts in “ProtectiveGroups in Organic Chemistry,” John Wiley and Sons, 1991. Detailedexamples for the synthesis of compounds 200-17, 200-73, 200-93(resolution of isomers), 200-115, 200-117, 200-123, 200-139, and 200-149can be found in the Examples.

Synthesis of 200-17: Compound 200-17 is synthesized using the methodshown in Scheme 1 starting with commercially available2,6-difluoro-3-nitrobenzoic acid (1) and converting it into thecorresponding acyl chloride (2). To a mixture of 5-bromo-7-azaindole in1,2-dichloroethane is added a Lewis Acid (i.e., aluminum chloride)followed by addition of the acyl chloride (2) to produce(5-bromo-1H-pyrrolo[2,3-b]pyridine-3-yl)-2,6-difluoro-3-nitrophenyl)methanone(3). The nitrophenyl group of intermediate (3) is reduced to form(3-amino-2,6-difluoro-phenyl)-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methanone(4) which is reacted under anhydrous conditions with2,6-dichlorodbenzoyl chloride in the presence of triethylamine and4-dimethylaminopyridine to produce(3-amino-2,6-difluoro-phenyl)-[5-bromo-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl] methanone (5). The intermediate (5) is reactedwith 4-chlorobenzeneboronic acid and palladium dichloride to produce(3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6). The intermediate (6) is reacted with3-(acetyloxy)-1-propanesulfonyl chloride to formN-[3-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]3-(acetyloxy)propane-1-sulfonamide (7). The intermediate (7) isdeprotected, removal of the dichlorobenzoyl group, using standardconditions to produce 200-17.

Synthesis of 200-73: Compound 200-73 is synthesized using the methodshown in Scheme 2 starting from the(3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) and reacting it withprop-2-ene-1-sulfonyl chloride (22) to produceN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl] prop-2-ene-1-sulfonamide (23).Intermediate (23) is oxidized (e.g. osmium tetroxide) to produce thediol derivativeN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]2,3-dihydroxypropane-1-sulfonamide (24). The protecting group, thedichlorobenzoyl, is removed using standard procedures to produce 200-73.

Synthesis of 200-93: Compound 200-93 is synthesized using the methoddescribed in Scheme 3 starting from(3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) and reacting it with2-(benzyloxy)-1-propanesulfonyl chloride (35) in dioxane containingpyridine and 4-dimethylaminopyridine to produceN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]prop-1-ene-1-sulfonamide (36).The intermediate (36) is deprotected, removal of the dichlorobenzoylgroup, using standard conditions to produce 200-93 as a mixture of (cis)and (trans) isomers. The two isomers are resolved by HPLC, and named200-93a and 200-93b, respectively.

Synthesis of 200-115: Compound 200-115 is synthesized using the methodshown in Scheme 4 starting from(3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) and reacting it with2-oxopropanesulfonyl chloride, to produceN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-oxopropane-1-sulfonamide(200-111). The intermediate (200-111) is treated sodium borohydride toreduce the ketone to the alcohol and produceN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-hydroxypropane-1-sulfonamide(200-113). The alcohol (200-113) is deprotected by removal of thedichlorobenzoyl group, using standard conditions to produce 200-115consisting of a mixture of R and S isomers.

Synthesis of 200-117: Compound 200-117 is synthesized using the methodshown in Scheme 5 starting fromN-[3-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-oxopropane-1-sulfonamide(200-111). The protecting dichlorobenzoyl group is removed usingstandard conditions to produce 200-117.

Synthesis of 200-123: Compound 200-123 is synthesized using the methodshown in Scheme 6 starting from3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) [see Scheme 1] and reacting it with3-methoxypropane-1-sulfonyl chloride (200-120) in dioxane containingpyridine and 4-dimethylaminopyridine to produceN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-3-methoxypropane-1-sulfonamide(200-121). The protecting dichlorobenzoyl group is removed usingstandard conditions to produce 200-123.

Synthesis of 200-129: Compound 200-139 is synthesized using the methodshown in Scheme 7 starting from3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) [see Scheme 1] and reacting it with2-methoxyethanesulfonyl chloride (200-136) in dioxane containingpyridine and 4-dimethylaminopyridine to produceN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-methoxyethanesulfonamide(200-137). The protecting dichlorobenzoyl group is removed usingstandard conditions to produce 200-139.

Synthesis of 200-149: Compound 200-149 is synthesized using the methodshown in Scheme 8 starting fromN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-methoxyethanesulfonamide(200-137) and reacting it with BBr₃ in DCM to produceN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-hydroxyethanesulfonamide(200-147). The protecting dichlorobenzoyl group is removed usingstandard conditions to produce 200-149.

EXAMPLES

Examples related to the present invention are described below. In mostcases, alternative techniques can be used. The examples are intended tobe illustrative and are not limiting or restrictive to the scope of theinvention. In most cases, alternative techniques can be used. In someexamples, the mass spectrometry results indicated for a compound mayhave more than one value due to the isotope distribution of an atom inthe molecule, such as a compound having a bromo or chloro substituent.

Example 1 Synthesis ofN-[3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-3-hydroxypropane-1-sulfonamide(200-17)

Compound 200-17 was synthesized by the procedure shown in Scheme 1.

Preparation of 2,6-difluoro-3-nitrobenzoyl chloride (2): To a 259 mLflask were added 2,6-difluoro-3-nitrobenzoic acid (8 g, 39.39 mmol),dichloromethane (80 mL), and DMF (0.3 mL). With stirring, oxalylchloride (10.4 mL, 118.81 mmol, 3 eq) was added dropwise at roomtemperature under nitrogen. After addition, the mixture was stirred atroom temperature under nitrogen overnight. The mixture was thenconcentrated under reduced pressure to get a solid residue, which wasdried under vacuum and used directly in next step.

Preparation of(5-bromo-IH-pyrrolo[2,3-b]pyridin-3-yl)-(2,6-difluoro-3-nitrophenyl)methanone(3): To a 500 mL, three-necked flask were added 5-bromo-7-azaindole(7.76 g, 39.38 mmol) and 1,2-dichloroethane (DCE, 120 mL). The mixturewas cooled to 0-5° C., and aluminum chloride (21 g, 157.49 mmol) wasadded in portions with stirring at 0-5° C. To the mixture was added amixture of 2,6-difluoro-3nitrobenzoyl chloride (2, about 39.39 mmol) andDCE (50 mL) dropwise. During the addition, the reaction temperature wasmaintained below 25° C. After the addition, the reaction mixture asstirred at room temperature for 30 min and at 50° C. under nitrogenovernight. TLC analysis (EtOAc-hexanes; 1:1) showed the reaction wascomplete. The mixture was cooled to room temperature and poured to coldwater (600 mL), followed by extraction with EtOAc (200 mL). The organiclayers were washed with brine, dried (Na₂SO₄) and concentrated to givecompound 3 as a yellow-brown solid, which was directly used in nextreduction. LC-MS data: 381.60 (M+H).

Preparation of(3-amino-2,6-difluoro-phenyl)-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methanone(4): To a 1 L flask were added compound 3 (about 39.39 mmol),tetrahydrofuran (THF, 150 mL), and tin(II) chloride (22.4 g, 118.13mmol, 3 eq). The mixture was heated to 60-65° C. under nitrogenovernight. TLC analysis (EtOAc-hexanes; 1.1) showed the reaction wascomplete. After the mixture was cooled to room temperature, EtOAc (200mL) was added, followed by slow addition of 10% NaHCO₃ solution withmuch gas being released. The resulting mixture was filtered through alayer of silica gel and the solid residue was washed with EtOAc. Theorganic of the filtrate was washed with brine, dried (Na₂SO₄) andconcentrated. The crude product was purified by silica gelchromatography (EtOAc-hexanes; 1:3 to 1:1) to give compound 4 as ared-brown solid (10.2 g, 74% yield over 3 steps). LC-MS data: 351.50(M+H).

Preparation of(3-amino-2,6-difluoro-phenyl)-[5-bromo-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl] methadones (5): To a 250 mL flask were addedcompound 4 (2.2 g, 6.25 mmol), THF (70 mL), triethylamine (1.2 mL, 8.61mmol, 1.3 eq), and 4-dimethylaminopyridine (DMAP) (40 mg). The mixturewas cooled to 0-5° C., then 2,6-dichlorobenzoyl chloride (0.9 mL, 6.28mmol) dropwise. After addition, the mixture was stirred at 0-5° C. for 1hrs when TLC analysis (EtOAc-hexanes, 1:2) showed no presence of thestarting material. The reaction was quenched with methanol (1 mL) at0-5° C. EtOAc (100 mL) and water (100 mL) were added. The organic layerwas separated, and the aqueous lay was extracted with EtOAc (100 mL).The combined organic layers were washed with brine, dried (Na₂SO₄), andconcentrated. The residue was subjected to column chromatography(EtOAc-hexanes; 1:10 to 1:5) to give compound 5 as a yellow solid, 1.6g, 49% yield), LC-MS data: 523.70 (M+H).

Preparation of(3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6); Compound 5 (900 mg, 1.71 mmol),4-chlorobenzeneboronic acid (374 mg, 2.39 mmol, 1.4 eq), potassiumcarbonate (475 mg, 3.44 mmol, 2 eq), dioxane (10 mL), and water (6 mL)were added to a 100 mL flask. The mixture was purged with nitrogen.PdCl₂ (dppf) (40 mg; 0.0546 mmol. 3% eq) was added and the reactionmixture was heated to 80-82° C. and stirred for 2 hrs. TLC and HPLCshowed the reaction was complete. The reaction mixture was cooled andfiltered over Celite and washed with EtOAc. The organic layer of thefiltrate was separated, washed with brine and water, dried (Na₂SO₄), andconcentrated. The residue was subjected to column chromatography(EtOAc-hexanes; 1:10 to 1:4) to give compound 6 as a yellow solid, 710mg, 75% yield). LC-MS data: 555.90 (M+H).

Preparation of sodium 3-(acetyloxy)-1-propanesulfonate (11): In a 100 mLflask were added sodium 3-hydroxy-1-propanesulfonate (5 g, 80% purity,24.67 mmol) and acetic anhydride (12 mL, 126.95 mmol, 5.1 eq). Themixture was heated at reflux under nitrogen for 7 hrs and then cooled toroom temperature. It was almost a solid. TBME (60 mL) was added, and theresultant mixture was stirred for 10 min, followed by filtration,washing with TBME and hexanes, and drying under vacuum to give compound11 as a white solid (7.8 g).

Preparation of 3-(acetyloxy)-1-propanesulfonyl chloride (12): In a 100mL flask was added sodium 3-(acetyloxy)-1-propanesulfonate (11, 4 g, ca19.59 mmol), followed by dropwise addition of thionyl chloride (8 mL,109.67 mmol, 5.6 eq). The mixture was then stirred at 68° C. undernitrogen for 5 hrs. The mixture was cooled and concentrated underreduced pressure to remove excess thionyl chloride. TBME (methyltert-butyl ether) was added, followed by filtration and washing withTBME. The filtrate and washing were concentrated, and the resultantliquid was dried under vacuum to afford compound 12 as a pale brownliquid (1.8 g), which was used in next step without purification.

Preparation ofN-[3-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]3-(acetyloxy)propane-1-sulfonamide (7): To a 100 mL flask were addedcompound 6 (700 mg, 1.26 mmol), dioxane (15 mL), pyridine (1 mL, 12.4mmol, 10 eq), and DMAP (20 mg), 3-(Acetyloxy)-1-propanesulfonyl chloride(12, 866 mg, 4.32 mmol, 3.4 eq) was added dropwise with stirring at roomtemperature. The reaction mixture is heated at 99-100° C. under nitrogenfor 4 hrs when TLC analysis (EtOAc-hexanes; 1:2) and HPLC showed thereaction was complete. The mixture was cooled, and EtOAc (100 mL) wasadded, followed by addition of water. The organic layer was separated,and the aqueous layer was extracted with EtOAc (2 times). The combinedorganic layers were washed with brine, dried (Na₂SO₄), and concentratedto give a liquid residue. The residue was subjected to columnchromatography (EtOAc-hexanes; 1:5 to 1:3) to give compound 7 as a solid(420 mg, 46% yield). LC-MS data: 720.20 (M+H).

Preparation ofN-[3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-3-hydroxypropane-1-sulfonamide(200-17): in a 100 mL flask were added compound 7 (420 mg) and methanol(5 mL). Ammonia solution (27%, 5 mL) was added dropwise with stirring.After addition, the mixture was stirred for 14 hrs at 33-35° C. TLCanalysis (EtOAc-hexanes; 1:1) showed the reaction was complete. Themixture was concentrated. EtOAc (20 mL) and water (20 mL) were added.The organic layer was separated, and the aqueous lay was extracted withEtOAc (20 mL). The combined organic layers were washed with brine, dried(Na₂SO₄), and concentrated. The residue was subjected to columnchromatography (DCM-MeOH; 95:5) to give compound 200-17 as a pale yellowsolid (150 mg, 51% yield), LC-MS data: 506.10 (M+H); ¹H NMR (DMSO-d₆)(ppm) 13.05 (s, broad, 1H), 9.78 (s, 1H), 8.71 (d, 1H), 8.64 (s, 1H),8.22 (s, 1H), 7.81 (d, 2H), 7.62-7.56 (m, 3H), 7.26 (t, 1H), 4.64 (,1H), 3.20 (m, 2H), 2.49 (m, 2H), 1.82 (m, 2H).

Example 2 Preparation of N-[3-[5-(4-chlorophenyl)-IH-1-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2,3-dihydroxypropane-I-sulfonamide(200-73)

Compound 200-73 was synthesized starting from(3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl] methanone (6) by the synthetic procedure shown inScheme 2.

Preparation of prop-2-ene-1-sulfonyl chloride (22): In a 200 flask wasadded sodium prop-2-ene-1-sulfonate (21, 4 g 27.75 mmol), followed bydropwise addition of thionyl chloride (10 mL, 137.09 mmol, 4.9 eq) atroom temperature. The mixture was then stirred at 65° C. under nitrogenfor 1 hrs. Due to stirring difficulty, benzene (5 mL) and thionylchloride (2 mL) were added. The mixture was stirred at 65° C. undernitrogen, overnight. After the mixture was cooled to room temperature,TBME (30 mL) and sodium sulfate were added. The mixture was stirred for30 min and filtered through a layer of silica gel. The filtrate wasconcentrated to give compound 22 as a pale brown liquid (2.95 g, 76yield), which was used in next step without purification.

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl] prop-2-ene-1-sulfonamide (23):To a 100 mL flask were added compound 6 (370 mg, 0.664 mmol), dioxane(15 mL), pyridine (0.5 mL, 6.18 mmol, 9.3 eq), and DMAP (20 mg).Prop-2-ene-1-sulfonyl chloride (22, 400 mg, 2.85 mmol, 4.3 eq) was addeddropwise with stirring at room temperature. The reaction mixture isheated at 85° C. under nitrogen for 2 hrs when TLC analysis(EtOAc-hexanes, 1:2) and HPLC showed the reaction was complete. Themixture was cooled, and EtOAc (100 mL) was added, followed by additionof water. The organic layer was separated, and the aqueous layer wasextracted with EtOAc (2 times). The combined organic layers were washedwith brine, dried (Na₂SO₄), and concentrated to give a liquid residue.The residue was subjected to column chromatography (EtOAc-hexanes; 1:5to 1:3) to give compound 23 as a yellow-brown solid (410 mg, 93% yield).LC-MS data: 659.90, 663.10 (M+H).

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]2,3-dihydroxypropane-1-sulfonamide (24): To a 100 mL flask were addedcompound 23 (500 mg, 0.757 mmol), acetone (24 mL), N-methylmorpholineN-oxide (178 mg, 1.52 mmol, 2 eq), and water (6 mL) at room temperature.Osmium tetroxide solution (4%, 4 mL) was added dropwise with stirring.The reaction mixture is stirred at room temperature overnight when TLCanalysis (EtOAc-hexanes; 1:2) showed the reaction was complete. EtOAc(100 mL) was added, followed by addition of water. The organic layer wasseparated, and the aqueous layer was extracted with EtOAc (2 times). Thecombined organic layers were washed with brine, dried (Na₂SO₄), andconcentrated to give a liquid residue. The residue was subjected tocolumn chromatography (EtOAc-hexanes; 2:1) to give compound 24 as a palebrown solid (300 mg, 57% yield). LC-MS data: 694.00, 696.10 (M+H).

Preparation ofN-[3-[5-(4-chlorophenyl)-IH-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2,3-dihydroxypropane-I-sulfonamide(200-73): In a 100 mL flask were added compound 24 (300 mg) and THF (2mL), and methanol (5 mL). Ammonia solution (27%, 5 mL) was addeddropwise with stirring. After addition, the mixture was stirred for 3hrs at 33-35° C. TLC analysis (EtOAc only) showed the reaction wascomplete. The mixture was concentrated. EtOAc (20 mL) and water (20 mL)were added. The organic layer was separated, and the aqueous lay wasextracted with EtOAc (20 mL). The combined organic layers were washedwith brine, dried (Na₂SO₄), and concentrated. The residue was subjectedto column chromatography (DCM-MeOH; 95:5) to give compound 200-73 as anoff-white solid (120 mg, 53% yield). LC-MS data: 521.80, 523.90 (M+H);¹H NMR (DMSO-d₆) (ppm) 13.00 (s, broad, 1H), 9.64 (s, 1H), 8.71 (d, 1H),8.65 (s, 1H), 8.19 (s, 1H), 7.81 (d, 2H), 7.62-7.56 (m, 3H), 7.26 (t,1H), 5.08 (d, 1H), 4.81 (t, 1H), 4.01 (m, 2H), 3.30 (m, 2H), 3.05 (m,1H).

Example 3 Preparation ofN-[3-[5-(4-chlorophenyl)-IH-1-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]prop-1-ene-I-sulfonamide(200-93)

Compound 200-93 a synthesized by the procedure shown in Scheme 3.

Preparation of 1-mercapto-2-(benzyloxy)propane (34): In a 250 mL flaskwere added propene oxide (2 g, 34.43 mmol) and thiobenzoic acid (4.76 g,34.44 mmol), by dropwise addition of triethylamine (0.4 mL, 2.86 mmol)at room temperature. It was observed that reaction occurred suddenlywhen 0.2 mL of triethylamine was added. After stirring at roomtemperature for 15 min, TLC showed reaction was complete. Silica gel (18g) was added. The mixture was then stirred at 65° C. under nitrogenovernight. TLC showed 2-hydroxythioester (33) was mainly converted to1-mercapto-2-benzyloxypropane. After the mixture was cooled to roomtemperature, TBME (50 mL) was added. The mixture was stirred for 30 minand filtered through a layer of silica gel. The filtrate wasconcentrated to give compound 34 as a pale brown liquid (5.8 g), whichwas used in next step without purification.

Preparation of 2-(benzyloxy)-1-propanesulfonyl chloride (35): NCS (5.44g, 40.74 mmol, 4 eq) was added to a mixture of 2N HCl solution and MeCN(8 mL/18 mL). The mixture was cooled to 10° C. A solution of 34 (2 g,10.19 mmol) was added dropwise below 20° C. After addition, the mixturewas stirred below 20° C. for 20 min. TBME (30 mL) was added. The organiclayer was separated and washed with 12% NaOH solution (3×8 mL), dried(Na₂SO₄), concentrated, and purified by column purification(EtOAc-hexanes; 1:6) to give compound 35 as a pale yellow liquid (3.8g).

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl] prop-1-ene-1-sulfonamide (36):To a 100 mL flask were added compound 6 (370 mg, 0.664 mmol), dioxane(15 mL), pyridine (0.5 mL, 6.18 mmol, 9.3 eq), and DMAP (20 mg).2-(Benzyloxy)-1-propanesulfonyl chloride (35, 700 mg, 2.66 mmol, 4 eq)was added dropwise with stirring at room temperature. The reactionmixture is heated at 85° C. under nitrogen for 8 hrs when TLC analysis(EtOAc-hexanes; 1:2) and HPLC showed the reaction was complete. Themixture was cooled, and EtOAc (100 mL) was added, followed by additionof water. The organic layer was separated, and the aqueous layer wasextracted with EtOAc (2 times). The combined organic layers were washedwith brine, dried (Na₂SO₄), and concentrated to give a liquid residue.The residue was subjected to column chromatography (EtOAc-hexanes; 1:5to 1:3) to give compound 36 as a pale brown solid (350 mg, 80% yield).LC-MS data: 659.90, 663.10 (M+H).

Preparation ofN-[3-[5-(4-chlorophenyl)-IH-1-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]prop-1-ene-I-sulfonamide (200-93): In a 100 mL flask were added compound24 (300 mg) and THF (2 mL), and methanol (5 mL). Ammonia solution (27%,5 mL) was added dropwise with stirring. After addition, the mixture wasstirred for 3 hrs at 33-35° C. TLC analysis (EtOAc only) showed thereaction was complete. The mixture was concentrated. EtOAc (20 mL) andwater (20 mL) were added. The organic layer was separated, and theaqueous lay was extracted with EtOAc (20 mL). The combined organiclayers were washed with brine, dried (Na₂SO₄), and concentrated. Theresidue was subjected to column chromatography (DCM-MeOH; 95:5) to givea mixture of two isomers (cis- and trans-) of compound 200-93 as a paleyellow brown solid (35 mg). LC-MS data: 488.0, 490.20 (M+H). 200-93isomers were further separated by HPLC on a YMC-Pack ODS column (100×20mm, 50 μm, 120 A) eluted with methanol (B) and water (A) in a stepwisegradient over 20 min running time. The two isomers were named as 200-93aand 200-93b with HPLC retention time of 11 and 9 min, respectively.

Example 4 Preparation ofN-[3-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-hydroxypropane-1-sulfonamide(00-115)

Compound 200-115 was synthesized starting from(3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) by the synthetic procedure shown inScheme 4.

Preparation of sodium 2-oxopropanesulfonate (200-105): Chloroacetone (8mL, 0.10 mol), Na₂SO₃ (14.5 g, 0.115 mol) and H₂O (100 mL) were mixed ina flask equipped with a condenser. The mixture was refluxed withstirring for 20 hrs, after which the mixture was evaporated to dryness.Ethanol (30 mL) was added to the residue, followed by concentration.TBME-hexanes (1:1; 30 mL) were added. After stirring a while,filtration, washing with hexanes, and drying under reduced pressure gavea white solid (28 g), which contained the product sodium2-oxopropanesulfonate and sodium chloride with about 70% purity.

Preparation of 2-Oxopropanesulfonyl chloride (200-110): To a mixture oftoluene (5 mL) and POCl₃ (5 mL) at 5° C. was added sodium2-oxopropanesulfonate (4 g, about 70% purity) in portions. The mixturewas then heated (oil bath 110° C.) for 3 hrs. The solvent was removed byevaporation, the product was dissolved in DCM (15 mL), and the mixturewas filtered. The filtrate was concentrated to yield the title compoundas a dark brown liquid (1.5 g), which was used without furtherpurification.

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-oxopropane-1-sulfonamide(200-111): To a 100 mL flask were added compound 6 (450 mg, 0.81 mmol),dioxane (15 mL), pyridine (0.6 mL, 7.42 mmol, 9.2 eq), and DMAP (30 mg).2-Oxopropanesulfonyl chloride (510 mg, 3.26 mmol, 4 eq) was addeddropwise with stirring at room temperature. The reaction mixture washeated at 85° C. under nitrogen for 2 hrs when TLC analysis(EtOAc-hexanes; 1:2) and HPLC showed the reaction was complete. Themixture was cooled, and EtOAc (100 mL) was added, followed by additionof water. The organic layer was separated, and the aqueous layer wasextracted with EtOAc (2 times). The combined organic layers were washedwith brine, dried (Na₂SO₄), and concentrated to give a liquid residue.The residue was subjected to column chromatography (EtOAc-hexanes: 1:5to 1:3) to give compound 200-111 as a pale brown solid (490 mg, 89%yield). LC-MS data; 676 (M+H).

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-hydroxypropane-1-sulfonamide(200-113): To a mixture of compound 200.111 (219 mg, 0.81 mmol) andmethanol (30 mL) and THF (5 mL) at 0° C. was added a solution of NaBH4(13 mg) in MeOH (5 mL) dropwise. After addition, the mixture was stirredat 0-5° C. for 10 min and TLC analysis (EtOAc-hexanes: 1:2) showed theabsence of the starting material. Water (2 mL) was, added to quench thereaction. EtOAc (100 mL) was added, followed by addition of water. Theorganic layer as separated, and the aqueous layer was extracted withEtOAc (2 times). The combined organic layers were washed with brine,dried (Na₂SO₄), and concentrated, to give a liquid residue. The residuewas subjected to column chromatography (EtOAc-hexanes; 1:4 to 1:2) togive 200-113 as an off-white solid (110 mg, 51%). LC-MS data: 678.2(M+H).

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-hydroxypropane-1-sulfonamide(200-115): In a 100 mL flask were added compound 200-113 (95 mg),methanol (5 mL), and THF (3 mL). Ammonia solution (27%, 4 mL) was addeddropwise with stirring. After addition, the mixture was stirred for 3hrs at 33-35° C. TLC analysis (EtOAc-hexanes; 1:1) showed the reactionwas complete, EtOAc and sodium sulfate (Na₂SO₄) were added. Afterstirring a while, the mixture was filtered, and the filtrate wasconcentrated. The residue was subjected to column chromatography(EtOAc-hexanes; 1:3, then EtOAc only) to give 200-115 as a pale brownsolid (48 mg) LC-MS data: 506.30 (M+H); H NMR (DMSO-d₆) (ppm) 13.02 (s,broad, 1H), 9.75 (s, 1H), 8.71 (d, 1H), 8.64 (s, 1H), 8.22 (s, 1H), 7.81(d, 2H), 7.62-7.56 (m, 3H), 7.26 (t, 1H), 5.10 (s, 1H), 4.10 (m, 1H),3.12 (m, 2H), 1.22 (d, 3H).

Example 5 Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-oxopropane-1-sulfonamide(200-117)

Compound 200-117 was synthesized starting fromN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-oxopropane-1-sulfonamide(200-111) by the synthetic procedure shown in Scheme 5.

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-2-hydroxypropane-1-sulfonamide(200-117): To a 100 mL flask were added compound 200-111 (110 mg),methanol (5 mL), and THF (3 mL). Ammonia solution (27%, 4 mL) was addeddropmise with stirring. After addition, the mixture was stirred for 3hrs at 33-35° C. TLC analysis (EtOAc-hexanes, 1:1) showed the reactionwas complete. EtOAc and sodium sulfate (Na₂SO₄) were added. Afterstirring a while, the mixture was filtered, and the filtrate wasconcentrated. The residue was subjected to column chromatography(EtOAc-hexanes; 1:3, then 2:1) to give 200-117 as an off-white solid (49mg). LC-MS data: 503.90 (M+H); ¹H NMR (DMSO-d₆) (ppm) 13.02 (s, broad,1H), 10.05 (s, 1H), 8.71 (d, 1H), 8.64 (s, 1H), 822 (s, 1H), 7.81 (d,2H), 7.62-7.56 (m, 3H), 7.26 (t, 1H), 4.40 (s, 2H), 2.22 (s, 3H).

Example 6 Preparation of N-[3-[5-(4-chlorophenyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-3-methoxypropane-1-sulfonamide(200-123)

Compound 200-123 was synthesized starting from3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) by the synthetic procedure shown inScheme 6.

Preparation of sodium 3-methoxypropane-1-sulfonate (200-119):1-Bromo-3-methoxypropane (7.5 g, 49.01 mmol), Na₂SO₃ (6.2 g, 49.19 mmol)and H₂O (50 mL) were mixed in a flask equipped with a condenser. Themixture was refluxed with stirring for 20 hrs, after which the mixturewas evaporated to dryness. Ethanol (30 mL) was added to the residuefollowed by concentration. TBME-hexanes (1:1, 30 mL) were added.Following stirring, filtration, washing with hexanes, and drying undervacuum gave a white solid (10.5 g), which contained the product sodium3-methoxypropane-1-sulfonate and sodium bromide with about 63% purity.

Preparation of 3-Methoxypropane-1-sulfonyl chloride (200-120): To amixture of toluene (16 mL) and thionyl chloride (10 mL) at 5° C. wasadded sodium 3-methoxypropane-1-sulfonate (200-119) (5 g, about 63%purity) in portions. The mixture was heated to 80° C. for 4 hrs. Themixture was cooled and concentrated, and the residue was dissolved inDCM (15 mL). The mixture as filtered and the filtrate concentrated toyield the 200-120 as a brown liquid (1.95 g), which was used withoutfurther purification.

Preparation ofN-[3-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl]-3-methoxypropane-1-sulfonamide(200-121): To a 100 mL flask were added compound 6 (250 mg, 0.45 mmol),dioxane (15 mL), pyridine (0.6 mL, 7.42 mmol, 16.4 eq), and DMAP (25mg). 3-methoxypropane-1-sulfonyl chloride (200-120) (200 mg, 1.16 mmol,2.6 eq) was added dropwise with stirring at room temperature. Thereaction mixture was heated at 85° C. under nitrogen for 6 hrs wherebyTLC (EtOAc-hexanes, 1:2) and HPLC showed the reaction to be complete.The mixture was cooled and EtOAc (100 mL) added followed by addition ofwater. The organic layer was separated and the aqueous layer extractedwith EtOAc (2 times). The combined organic layers were washed withbrine, dried with Na₂SO₄, and concentrated to give a liquid residue. Theresidue was subjected to column chromatography (EtOAc-hexanes, 1:6 to1:3) to give compound 200-121 as a pale brown solid (150 mg, 48% yield).LC-MS data. 682.00 (M+H).

Preparation of N-[3-[5-(4-chlorophenyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-3-methoxypropane-1-sulfonamide(200-123): In a 100 mL flask were added compound 200-121 (150 mg, 0.22mmol), methanol (4 mL), and THF (2 mL). Ammonia solution (27%, 4 mL) wasadded dropwise with stirring. After addition, the mixture was stirredfor 3 hrs at 33-35° C. TLC analysis (EtOAc-hexanes, 1:1) showed thereaction to be complete. EtOAc and sodium sulfate (Na₂SO₄) were added tothe mixture. After stirring a period of time, the mixture was filteredand the filtrate concentrated. The residue was subjected to columnchromatography (EtOAc-hexanes, 1:2, then 2:1) to give compound 200-123as an off-white solid (48 mg). LC-MS data: 520.00 (M+H): ¹H NMR(DMSO-d₆) (ppm) 13.02 (s, broad, 1H), 9.85 (s, 1H), 8.71 (s, 1H), 8.64(d, 1H), 8.22 (s, 1H), 7.81 (d, 2H), 7.62-7.56 (m, 3H), 7.26 (t, 1H),3.40 (t, 2H), 3.56 (s, 3H), 1.95 (m, 2H). 1.20 (t, 2H).

Example 7 Preparation of N-[3-[5-(4-chlorophenyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-methoxyethanesulfonamide(200-139)

Compound 200-139 was synthesized starting from3-amino-2,6-difluorophenyl)-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b] pyridin-3-yl]methanone (6) by the synthetic procedure shown inScheme 7.

Preparation of sodium 2-methoxyethanesulfonate (200-135):1-Bromo-2-methoxyethane (7.5 g, 53.96 mmol), Na₂SO₃ (6.8 g, 53.95 mmol)and H₂O (50 mL) were mixed in a flask equipped with a condenser. Themixture was refluxed with stirring for 20 hrs. The mixture wasevaporated to dryness. Ethanol (30 mL) was added to the residue,followed by concentration. TBME-hexanes (1:1, 40 mL) were added. Afterstirring for a period of time, filtration, washing with hexanes, anddrying under vacuum gave white solid (14 g) comprising the productsodium 2-methoxyethanesulfonate (200-135) (61% purity) and sodiumbromide.

Preparation of 2-Methoxyethanesulfonyl chloride (200-136): To a mixtureof thionyl chloride (15 mL) at 5° C. was added sodium2-methoxyethanesulfonate (200-135) (5 g, about 61% purity) in portions,followed by addition of 3 drops of DMF. The mixture was stirred at roomtemperature for 20 min followed by heating at 80° C. under nitrogen for6 hrs. The mixture was cooled and concentrated, and the residue wasdissolved in TBME (15 mL). The mixture was filtered and the filtrateconcentrated to yield the title compound 200-136 as a pale brown liquid(1.85 g), which was used without further purification.

Preparation ofN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-methoxyethanesulfonamide(200-137): To a 100 mL flask were added compound 6 (95 mg, 0.17 mmol),dioxane (15 mL), pyridine (0.5 mL), and DMAP (25 mg).2-methoxyethanesulfonyl chloride (200-136) (100 mg, 0.63 mmol, 3.7 eq)was added dropwise with stirring at room temperature. The resultantreaction mixture was heated at 85° C. under nitrogen for 6 hrs where TLC(EtOAc-hexanes, 1:2) and HPLC showed the reaction to be complete. Themixture was cooled and EtOAc (100 mL) added followed by addition ofwater. The organic layer was separated, and the aqueous layer extractedwith EtOAc (twice). The combined organic layers were washed with brine,dried with Na₂SO₄, and concentrated to give a liquid residue. Theresidue was purified by column chromatography (EtOAc-hexanes, 1:6 to1:3) to give compound 200-137 as a pale brown solid (59 mg, 51% yield).LC-MS data: 678.00 (M+H).

Preparation of N-[3-[5-(4-chlorophenyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-methoxyethanesulfonamide(200-139): In a 100 mL flask were added compound 200-137 (50 mg, 0.074mmol), methanol (5 mL), and THF (1 mL). Ammonia solution (27%, 2 mL) wasadded dropwise with stirring. After addition, the mixture was stirredfor 3 hrs at 33-35° C. and left to stir overnight at room temperature.TLC analysis (EtOAc-hexanes, 1:1) showed the reaction was complete.EtOAc and sodium sulfate (Na₂SO₄) were added. After stirring for aperiod of time, the mixture was filtered and the filtrate concentrated.The residue was purified by column chromatography (EtOAc-hexanes, 1:2,then 3:1) to give compound 200-139 as a pale yellow-brown solid (28 mg).LC-MS data: 506.00 (M+H); ¹H NMR (DMSO-d₆) (ppm) 13.02 (s, broad, 1H),8.71 (s, 1H), 8.64 (s, 1H), 8.22 (s, 1H), 7.81 (d, 2H), 7.62-7.56 (m,3H), 7.26 (t, 1H), 3.60 (t, 2H), 3.36 (s, 3H), 2.42 (t, 2H).

Example 8 Preparation of N-[3-[5-(4-chlorophenyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-hydroxyethanesulfonamide(200-149)

Compound 200-149 was synthesized starting fromN-[3-[5-(4-chlorophenyl)-I-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-methoxyethanesulfonamide(200-137) by the synthetic procedure shown in Scheme 8.

Preparation ofN-[3-[5-(4-chlorophenyl)-1-(2,6-dichlorobenzoyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-hydroxyethanesulfonamide(200-147): A solution of compound 200-137 (250 mg, 0.368 mmol) in DCMwas cooled to −64° C., BBr₃ (0.2 mL, 2.11 mmol) was added dropwise withstirring. Following addition, the mixture was stirred for 15 min at −64°C. and for 3 hrs at room temperature. EtOAc (50 mL) was added followedby addition of cold water. The organic layer was separated and theaqueous layer extracted with EtOAc (twice). The combined organic layerswere washed with brine, dried with Na₂SO₄, and concentrated to give aliquid residue. The residue eras purified by column chromatography(EtOAc-hexanes, 1:3 to 1:1) to give compound 200-147 as a pale brown oil(120 mg, 49% yield). LC-MS data: 664.17 (M+H).

Preparation of N-[3-[5-(4-chlorophenyl)pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-2-hydroxyethanesulfonamide(200-149): In a 100 mL flask were added compound 200-147 (120 mg, 0.18mmol), methanol (5 mL), and THF (2 mL). Ammonia solution (27%, 2 mL) wasadded dropwise with stirring. After addition, the mixture was stirredfor 2 hrs at 33-35° C. TLC analysis (EtOAc-hexanes, 1:1) showed thereaction was complete. EtOAc and sodium sulfate (Na₂SO₄) were added.After stirring for a period of time, the mixture was filtered and thefiltrate concentrated. The residue was purified by column chromatography(EtOAc-hexanes, 1:2, then DCM-MeOH, 20:1) to give compound 200-149 as anoff-white solid (58 mg) LC-MS data: 492.3 (M+H): ¹H NMR (DMSO-d₆) (ppm)13.02 (s, broad, 1H), 9.78 (s, 1H), 8.71 (s, 1H), 8.64 (s, 1H), 8.22 (s,1H), 7.81 (d 2H), 7.62-7.56 (m, 3H), 7.26 (t, 1H), 5.01 (s, 1H), 3.80(t, 2H), 2.50 (t, 2H).

Example 9 Protein Kinase Inhibition Studies

Off-chip Mobility Shift Assay (MSA) by Carna Biosciences, Inc (Natick,Mass.) was used for measuring the kinase activity and inhibition.

-   -   1) The 5 μL of ×4 compound solution, 5 μL of ×4        Substrate/ATP/Metal solution, and 10 μL of ×2 kinase solution        were prepared with assay buffer (20 mM HEPES, 0.01% Triton        X-100, 2 mM DTT, pH 7.5) and mixed, and incubated in a well of        polypropylene 384 well microplate for 1 or 5 hour(s)* at room        temperature. (*; depending on kinase)    -   2) 70 μL of Termination Buffer (QuickScout Screening Assist MSA;        Carna Biosciences) was added to the well.    -   3) The reaction mixture was applied to LabChip system (Perkin        Elmer), and the product and substrate peptide peaks were        separated and quantitated.    -   4) The kinase reaction was evaluated by the product ratio        calculated from peak heights of product (P) and substrate (S)        peptides (P/(P+S)).    -   5) The reaction conditions were followed according to assay        protocols of Carna Biosciences, Inc (BMA 3F, 1-5-5        Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan;        www.carnabio.com).    -   6) Data analysis: The readout value of reaction control        (complete reaction mixture) was set as a 0% inhibition, and the        readout value of background (Enzyme (−)) was set as 100%        inhibition, then the percent inhibition of each test solution        was calculated.

TABLE 1 Inhibition of kinase activities by compound 200-17 InhibitorInhibition concentration category Kinases 1 μM >50% BRK, FGR,PDGFRα(V561D), DDR2, inhibition LYNa, SRM, PDGFRα, LCK, DDR1, KDR, ACK,JAK1, LYNb, KIT, CSK, YES, KIT(V560G), BLK, MST1, JAK2, RET(S891A), SRC,FYN(isoform a), RET(G691S), FYN(isoform b), PDGFRβ, RET, FLT4,RET(Y791F), skMLCK, FRK, MST2, FLT1, AurA, FLT3, JAK3, RET(M918T), WNK3,p38β, FGFR2, MNK1, MNK2, PIK3CA/PIK3R1, PDGFRα(D842V), MET, FGFR1,BRAF(V600E), MAP2K5, KIT(D816E), ALK, FGFR3, RAF1, MAP2K3, HER4,KIT(D816V), Erk5, EGFR, YES(T348I), KIT(V654A) <50% KIT(D816Y),PDGFRα(T674I), BRAF, inhibition ABL, HER2, EPHA5, ROCK2, KIT(T670I),PKD3, MST4, MAP2K1, MAP2K2, MST3, ROCK1, IGF1R, PKD2, MAP2K6, Erk2,PKD1, MAP2K7, Erk1, MAP2K4, BTK 0.1 μM >50% SRM, BRK, FGR, LCK, JAK1,CSK, inhibition PDGFRα, DDR2, PDGFRα(V561D), MST1, KIT, YES, LYNa, DDR1,LYNb, RET(S891A), BLK, KDR, SRC, JAK2, KIT(V560G) <50% ACK, RET,FYN(isoform a), MST2, inhibition RET(G691S), RET(Y791F), PDGFRβ, JAK3,FYN(isoform b), RET(M918T), BTK 0.01 μM >30% SRM, BRK, FGR, LCK, KIT,JAK1 inhibition <30% PDGFRα, CSK, PDGFRα(V561D), YES, inhibitionRET(S891A), LYNa, DDR1, LYNb, BLK, RET, SRC, DDR2, KDR, RET(G691S),RET(M918T), MST1, JAK3, ACK, MST2, PDGFRβ, RET(Y791F), KIT(V560G),FYN(isoform a), FYN(isoform b), JAK2, BTK

TABLE 2 Inhibition of kinase activities by compounds 200-73, 200-93a,200-93b, 200-115, 200-117, 200-123, 200-139 Inhibition Inhibitorcategory Kinase 200-73 at >30% SRM, RAF1, JAK1, BRK 0.1 μM inhibition<30% LCK, KIT FGR, BRAF, MNK2, MAPKAPK2, inhibition MNK1, PIK3CA/PIK3R1,PDGFRα, LYNa, JAK3, BRAF(V600E), FLT3, JAK2, AurA, EGFR, LYNb, BTK,MLK1, KIT(V560G), HER2, SRC, ABL, PDGFRβ, Erk5, KIT(T670I)), MAP3K4,ROS, BMX, EGFR(L858R), IGF1R, MET, HER4, AXL, AKT2, RET, RON, PIM2,FGFR3, KIT(D816V), ABL(T315I), PIM3, JNK1, MAP2K1, AKT3, ABL(E255K),AKT1, KDR, ALK, PIM1, JNK2, JNK3, Erk2, Erk1, AurB, PKACα, MAP2K2, FAK,FGFR2 200-115 at >30% SRM, BRK, RAF1, PDGFRα, FGR, LCK 0.1 μM inhibition<30% LYNa, KIT, BRAF(V600E), JAK1, LYNb, inhibition MNK2, SRC,PIK3CA/PIK3R1, KDR, EGFR, MLK1, RON, MAPKAPK2, PDGFRβ, KIT(V560G), ABL,FLT3, KIT(T670I), HER2, MNK1, MET, MAP3K4, AKT2, AurA, BMX, JAK2, ROS,PIM2, AXL, EGFR(L858R), KIT(D816V), RET, AurB, FGFR3, HER4, IGF1R, JAK3,BTK, ABL(T315I), FGFR2, Erk5, AKT1, JNK1, AKT3, MAP2K1, ALK, BRAF,ABL(E255K), JNK3, PIM3, JNK2, PKACα, Erk2, FAK, Erk1, MAP2K2, PIM1200-117 at >30% BRK, SRM, LCK, FGR, RAF1, LYNa, 0.1 μM inhibitionPDGFRα, LYNb, MNK2 <30% KIT, BRAF(V600E), JAK1, SRC, inhibitionPIK3CA/PIK3R1, KDR, EGFR, MLK1, RON, MAPKAPK2, PDGFRβ, KIT(V560G), ABL,FLT3, KIT(T670I), HER2, MNK1, MET, MAP3K4, AKT2, AurA, BMX, JAK2, ROS,PIM2, AXL, EGFR(L858R), KIT(D816V), RET, AurB, FGFR3, HER4, IGF1R, JAK3,BTK, ABL(T315I), FGFR2, Erk5, AKT1, JNK1, AKT3, MAP2K1, ALK, BRAF,ABL(E255K), JNK3, PIM3, JNK2, PKACα, ErK2, FAK, Erk1, MAP2K2, PIM1200-93a at >20% PDGFRα, PDGFRβ 0.01 μM inhibition >30% PDGFRα(V561D),KIT(V560G), BRK, inhibition JAK1, KIT, PDGFRα(T674I), LCK, SRM, SRC,PDGFRα(D842V), FGR, KIT(T670I), JAK3, BRAF(V600E), JAK2, RAF1, BRAF,KIT(D816V) 200-93b at >20% SRM, BRK, FGR, RAF1, LCK, PDGFRα 0.01 μMinhibition <20% SRC, PDGFRα(V561D), BRAF(V600E), inhibition JAK1,PDGFRβ, PDGFRα(D842V), PDGFRα(T674I), JAK3, KIT(V560G), KIT KIT(T670I),BRAF, JAK2, KIT(D816V) 200-123 at >20% SRM, BRK, RAF1, BRAF(V600E), LCK0.01 μM inhibition <20% PDGFRα, JAK1, SRC, PDGFRα(V561D), inhibitionFGR, PDGFRβ, PDGFRα(T674I), PDGFRα(D842V), KIT(V560G), JAK3, KIT(T670I),KIT, JAK2, KIT(D816V), BRAF 200-139 at >20% SRM, BRK, RAF1, LCK, PDGFRα0.01 μM inhibition <20% JAK1, PDGFRα(V561D), FGR, PDGFRβ, inhibitionSRC, KIT(V560G), PDGFRα(D842V), BRAF(V600E), PDGFRα(T674I), KIT,KIT(T670I), JAK3, JAK2, KIT(D816V), BRAF

Example 10

Cell growth assay with breast cancer cell line T-47D: T-47D (ATCC®HTB-133™) was purchased from American Type Culture Collection (ATCC,Manassas, Va.). T-47D cells grew in DMDM medium (Gibco, LifeTechnologies) supplemented with 10% FBS (Gibco, Life Technologies)(complete medium) a T-75 flask at 37° C. under 5% CO₂ with saturatedhumidity. When the cells were approximately 70-80% confluent, theculture medium was removed, the cell layer was rinsed with 10 mLDulbecco's Phosphate-Buffered Saline (DPBS), and immediately followingthe treatment with 1 mL of 025% (w/v) Trypsin-EDTA solution at 37° C.for 5-15 min. A 9 mL aliquot of the complete medium was then gentlydispersed over the surface of the cell layer for several times. The cellconcentration was adjusted to 1×10⁴ cells/mL with the complete medium. A100 μL aliquot of the cell suspension was added to the well of a 96-wellplate, and the plate was incubated overnight at 37° C. under 5% CO₂ withsaturated humidity. On the next day, the medium in each well wasaspirated and replaced with a 100 μL aliquot of pre-warmed completemedium at 37° C. in the presence or absence of the test compounds atvarious concentrations from 0-100 μM. The test compounds were dissolvedin DMSO, and the final DMSO concentration in the cell culture was nomore than 1%. The plate was incubated for 72 hours at 37° C. under 5%CO₂ with saturated humidity. At end of the cell culture, a 10 μL aliquotof PrestoBlue® Cell Viability reagent (ThermoFisher Scientific) wasadded into the well, and the plate was incubated at 37° C. for 30 min.The absorption at 570 and 600 nm were measured with a SpectraMaxMicroplate reader (Molecular Devices). The absorbance at 570 nm wasnormalized to that at 600 nm. The normalized absorbance at 570 nm wasused for IC₅₀ calculation following the median-effect plot method (T. C.Chou, Pharmacol Rev 2006, 58: 621-681). The IC₅₀ values for 200-17,200-73, 200-93b, 200-115, 200-117, 200-123, 200-139 and 200-149 wereless than 20 μM, while the IC₅₀ value for 200-93a was greater than 20μM.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of appended claim. All publications, patents, and patentapplications cited herein are hereby incorporated by reference.

We claim:
 1. A compound of the following formula IV:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof; R¹ isa substituted lower alkyl or lower alkenyl, wherein the lower alkyl orlower alkenyl is substituted with one or more substituents selected from—OH, ═O, and alkoxy.
 2. The compound of claim 1, wherein said compoundis a compound of structure 200-17:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof. 3.The compound of claim 1, wherein said compound is a compound ofstructure 200-73:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof. 4.The compound of claim 1, wherein said compound is a compound ofstructure 200-115:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof. 5.The compound of claim 1, wherein said compound is a compound ofstructure 200-117:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof. 6.The compound of claim 1, wherein said compound is a compound ofstructure 200-123:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof. 7.The compound of claim 1, wherein said compound is a compound ofstructure 200-139:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof. 8.The compound of claim 1, wherein said compound is a compound ofstructure 200-149:

or a pharmaceutically acceptable salt, prodrug, or isomer thereof.